Gene expression patterns, including changes in gene expression between normal and diseased tissues or tissues in various stages of disease progression provide valuable insight into the molecular determinants of normal and abnormal cellular physiology. Accordingly, genes that are differentially expressed in subjects suffering from a disease, such as cardiac, renal or inflammatory disease, relative to normal subjects, are useful targets for intervention to diagnose, prevent or treat such diseases.
Techniques have been developed to efficiently analyze the level of expression of specific genes in cells and tissues. Procedures that can be used to identify and clone differentially expressed genes include, for example, subtractive hybridization (Jiang and Fisher, Mol. Cell. Different. (1993) 1:285–299; Jiang, et al., Oncogene (1995) 10:1855–1864; Sagerstrom, et al., Annu. Rev. Biochem. (1997) 66:751–83); differential RNA display (DDRT-PCR) (Watson, et al., Developmental Neuroscience (1993) 15:77–86; Liang and Pardee, Science (1992) 257:967–971); RNA fingerprinting by arbitrarily primed PCR (RAP-PCR) (Ralph, et al., Proc. Natl. Acad. Sci. USA (1993) 90:10710–10714; McClelland and Welsh, PCR Methods and Applications (1994) 4:S66–81); representational difference analysis (RDA) (Hubank and Schatz, Nucl. Acids Res. (1994) 22:5640–5648); serial analysis of gene expression (SAGE) (Velculescu, et al., Science (1995) 270:484–487; Zhang, et al., Science (1997) 276:1268–1272); electronic subtraction (Wan, et al., Nature Biotechnology (1996) 14:1685–1691); combinatorial gene matrix analyses (Schena, et al., Science (1995) 270:467–470), and various modifications and improvements of these and similar techniques.
A particularly attractive method for assessing gene expression is the DNA microarray technique. In this method, nucleotide sequences of interest are plated, or arrayed, on a porous or non-porous substrate that can be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support. The arrayed sequences are then hybridized with specific DNA probes from cells or tissues of interest. Microarrays of biological materials have been described in a number of patents and patent applications, including, for example, U.S. Pat. Nos. 5,744,305; 5,800,992; 5,807,522; and 5,716,785; and European Patent No. 0 373 203.
The DNA microarray technique can be used to monitor the expression level of large numbers of genes simultaneously (to produce a transcript image), and to identify genetic variants, mutations and polymorphisms. This information may be used to determine gene function, understanding the genetic basis of disease, diagnosing disease, and developing and monitoring the activities of therapeutic agents.
An important application of the microarray method allows for the assessment of differential gene expression in pairs of mRNA samples from two different tissues, or in the same tissue comparing normal versus disease states or time progression of the disease. Microarray analysis allows one to analyze the expression of known genes of interest, or to discover novel genes expressed differentially in tissues of interest. Thus, an attractive application of this technology is as a fundamental discovery tool to identify new genes, and their corresponding expression products, which contribute to the pathogenesis of disease and related conditions.
Microarray technology has been successfully applied to large-scale analysis of human gene expression to identify cancer-specific genes and inflammatory-specific genes (DeRisi, et al., Nat. Genet. (1996) 14(4):457–460; Heller, et al, Proc. Natl. Acad. Sci. USA (1997) 94(6):2150–2155). DeRisi, et al., examined a pre-selected set of 870 different genes for their expression in a melanoma cell line and a non-tumorigenic version of the same cell line. The microarray analysis revealed a decrease in expression for 15/870 (1.7%) and an increase in expression for 63/870 (7.3%) of the genes in non-tumorigenic relative to tumorigenic cells (differential expression values <0.52 or >2.4 were deemed significant). Heller, et al., employed microarrays to evaluate the expression of 1,000 genes in cells taken from normal and inflamed human tissues. The results indicated that altered expression was evident in genes encoding inflammatory mediators such as IL-3, and a tissue metalloprotease. These results illustrate the utility of applying microarray technology to complex human diseases.
It would be beneficial to discover differentially expressed genes that are related to diseases or various disease states. It would further be beneficial to develop methods and compositions for the diagnostic evaluation and prognosis of conditions involving such diseases, for the identification of subjects exhibiting a predisposition to such conditions, for modulating the effect of these differentially expressed genes and their expression products, for monitoring patients undergoing clinical evaluation for the prevention and treatment of a disease, specifically cardiac, kidney or inflammatory disease, and for monitoring the efficacy of compounds used in clinical trials.
Secreted proteins mediate key biological processes including cell to cell interactions as well as important cellular functions such as cell growth and differentiation, and most protein-based drugs are secreted proteins including insulin, growth hormone, interferons, tissue plasminogen activator (tPA), and erythropoietin (EPO). It would, therefore, be particularly desirable to identify novel differentially expressed genes encoding secreted proteins.